Intestinal peptide targeting ligands

ABSTRACT

Peptide ligands for transporting therapeutic agents across the intestinal epithelial barrier that ordinarily are inadequately absorbed and must be delivered by alternative means, which contain an isolated amino acid sequence wherein at least one pair of amino acids are of an opposite charge and the pair members are separated by a spacer of 1-12 amino acid residues including at least one hydrophobic amino acid, and wherein the length of the amino acid sequence is greater than 5 and less than 20 amino acids. Pharmaceutical compositions for gastro-intestinal delivery and methods for the gastrointestinal delivery of poorly absorbed therapeutic agents are also disclosed.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is the U.S. National Phase of International PatentApplication Serial No. PCT/US11/56449, filed on Oct. 14, 2011, whichclaims priority to U.S. Provisional Patent Application Ser. No.61/393,072, filed on Oct. 14, 2010, the disclosures of which areincorporated herein by reference in their entireties.

GOVERNMENT SUPPORT

This invention was made with government support under AI051214 awardedby the National Institutes of Health. The federal government has certainrights in this invention.

FIELD OF THE INVENTION

The present invention relates to peptide ligands that facilitatetransport of agents and carriers across the intestinal epithelialbarrier, which are useful for, inter alia, improving oral delivery andintestinal absorption of therapeutic, biologic and diagnostic agents.

BACKGROUND OF THE INVENTION

Oral delivery of therapeutic macromolecular drugs such as peptides,proteins (e.g. insulin) and siRNAs, as well as many small moleculeswithout natural intestinal carriers, has been challenging. While suchdrugs offer promising therapeutic value, when orally administered manyfail to be adequately transported from the patient's gastrointestinaltract to the bloodstream. Thus, very little of the drug is actuallyabsorbed and bioavailable for its intended therapeutic purpose.

As a result, there is a continuing need in the art for novel mechanismsof achieving increased drug absorption and bioavailability in the oraldelivery context. This is particularly true with macromolecular drugsand other known biologics.

SUMMARY OF THE INVENTION

The instant invention addresses the foregoing need. Peptide ligands havenow been discovered that transport therapeutic agents across theintestinal epithelial barrier that ordinarily are inadequately absorbedand must be delivered by alternative means. Accordingly, one aspect ofthe present invention provides peptide ligands with an amino acidsequence having at least one pair of amino acids of opposite charge,wherein the members of the pair are separated by a spacer of 1-12 aminoacid residues having at least one hydrophobic amino acid.

In one embodiment, the length of the amino acid sequence is greater than5 and less than 20 amino acids. In another embodiment, each pair ofamino acids of opposite charge include a negatively charged first memberselected from aspartic acid (D) or glutamic acid (E), and a positivelycharged second member selected from lysine (K), arginine (R), orhistidine (H). In another embodiment, the peptide ligand contains morethan one pair of amino acids of opposite charge. The pairs need not beidentical. In embodiments where multiple pairs of amino acids areprovided, such multiple pairs may be in juxtaposition or nested in thepeptide sequence.

In another embodiment, the pair(s) of oppositely charged amino acids areflanked with one or more non-charged or hydrophobic amino acids orsequences of non-charged amino acids, hydrophobic amino acids orcombinations thereof. Such amino acids may be provided at the N-terminusand/or C-terminus of the oppositely charged amino acid pair or thepeptide ligand sequence.

In a further embodiment the peptide ligand is coupled to a second aminoacid sequence

One embodiment of the peptide ligands of the instant invention may berepresented by the amino acid sequence of Formula 1:

X₁B₁Z₁Z₂Z₃B₂, wherein X₁ is not a charged or hydrophobic amino acid,Z₁-Z₃ is a spacer sequence of at least one hydrophobic amino acid, andB₁ and B₂ are members of the amino acid pair having opposite charge. Onenon-limiting example of such an amino acid sequence includes, but is notlimited to, TKWPVD (SEQ ID NO: 4). The peptide ligands of the instantinvention may also include variants or homologues of SEQ ID NO: 4, whichmay be at least 75% identical to SEQ ID NO: 4 or which may containconservative substitutions of corresponding amino acid residues.

In an alternative embodiment, the peptide ligands of the instantinvention may be represented by the amino acid sequence of Formula 2:X₁B₁Z₁Z₂Z₃B₂X₂X₃X₄,

wherein X₁-X₄ are not charged or hydrophobic amino acids, Z₁-Z₃ is aspacer sequence of at least one hydrophobic amino acid, and B₁ and B₂are members of the amino acid pair having opposite charge. Onenon-limiting example of such an amino acid sequence includes, but is notlimited to, TKWPVDMCP (SEQ ID NO: 5). The peptide ligands of the instantinvention may also include variants or homologues of SEQ ID NO: 5, whichmay be at least 75% identical to SEQ ID NO: 5 or which may containconservative substitutions of corresponding amino acid residues.

Another embodiment of the peptide ligands of the instant invention maybe represented by the amino acid sequence of Formula 3:X₁B₁Z₁Z₂Z₃B₂X₂X₃X₄X₅X₆X₇,

wherein X₁-X₇ represent uncharged or hydrophobic amino acids, Z₁-Z₃represent the spacer sequence of at least one hydrophobic amino acid,and B₁ and B₂ represent members of the amino acid pair having oppositecharges. One non-limiting example of such an amino acid sequenceincludes, but is not limited to, TKWPVDMCPNVS (SEQ ID NO: 1). Thepeptide ligands of the instant invention may also include variants orhomologues of SEQ ID NO: 1, which may be at least 75% identical to SEQID NO: 1 or which may contain conservative substitutions ofcorresponding amino acid residues.

In an alternative embodiment, the peptide ligands of the instantinvention may be represented by the amino acid sequence of Formula 4:X₁B₁B₂Z₁Z₂Z₃Z₄Z₅B₃Z₆Z₇B₄,

wherein X₁ represents an uncharged or hydrophobic amino acid and Z₁-Z₇represent the spacer sequence of at least one hydrophobic amino acid.B₁-B₄ represent the charged amino acids, wherein B₁ and B₂ have anidentical charge which is opposite to that of B₃ and B₄. Onenon-limiting example of such an amino acid sequence includes, but is notlimited to, QDDVQTWQRQPK (SEQ ID NO: 2). The peptide ligands of theinstant invention may also include variants or homologues of SEQ ID NO:2, which may be at least 75% identical to SEQ ID NO: 2 or which maycontain conservative substitutions of corresponding amino acid residues.

In an alternative embodiment, the peptide ligands of the instantinvention may be represented by the amino acid sequence of Formula 5:X₁B₁Z₁Z₂B₂Z₃B₃Z₄B₄Z₅Z₆B₅Z₇B₆X₂X₃X₄,

wherein X₁-X₄ represent uncharged or hydrophobic amino acids and Z₁-Z₇represent the spacer sequence of at least one hydrophobic amino acid.B₁-B₆ represent charged amino acids, wherein B₁, B₂ and B₃ have anidentical charge which is opposite to that of B₄, B₅ and B₆. Oneembodiment of such an amino acid sequence includes, but is not limitedto, GENFEQDWKSLRPHSSN (SEQ ID NO: 3). The peptide ligands of the instantinvention may also include variants or homologues of SEQ ID NO: 3, whichmay be at least 75% identical to SEQ ID NO: 3 or which may containconservative substitutions of corresponding amino acid residues.

The peptide ligands of the present make possible the delivery oftherapeutic agents across the intestinal epithelial barrier thatotherwise have inadequate gastrointestinal absorbtion. The peptideligands may be bound to the therapeutic agent or bound to a carriermolecule for co-delivery with a therapeutic agent. Accordingly, anotheraspect of the present invention provides a ligand peptide of the presentinvention bound to a therapeutic agent or a carrier for transport acrossthe intestinal epithelial barrier. In a further embodiment, more thanone peptide ligand is bound to a therapeutic agent or carrier. In afurther embodiment peptide ligands may be conjugated to a linker that isbound to a therapeutic agent or a carrier.

In another embodiment, the ligand peptide is bound to both a therapeuticagent and a carrier. In a further embodiment of the invention, multiplepeptide ligands may be bound to both a carrier and a therapeutic agent.In a further embodiment, multiple types of therapeutic agents may bebound to the carrier, for example a diagnostic agent, a targeting agent,an adjuvant, and multiple various drugs or other pharmaceutically activetherapeutic agents are bound to a carrier, as well as variant peptideligands. In a further embodiment agents and peptide ligands may be boundto a common carrier molecule by respective linkers. Agents may be boundto the surface of the carrier or located within the structure of thecarrier molecule.

In another embodiment, the present invention provides for a carrier thatis a nanoparticle or a microparticle.

In another embodiment, the carrier is a pharmaceutically acceptablepolymer for gastrointestinal drug delivery. In a further embodiment, thepolymer is polyethylene glycol.

In another embodiment, the present invention provides for a carrier thatis greater than 50 nm in diameter.

The present invention makes possible the gastrointestinal delivery of atherapeutic agent to a patient in need thereof across the intestinalepithelial barrier that otherwise is poorly absorbed gastrointestinally.Stated another way, the present invention provides a method ofdelivering a composition from an intestinal lumen across an intestinalepithelium of a subject. Such peptides facilitate the transport of thedrug across intestinal epithelial barrier in a chylomicron-forminglipid-dependent manner. Accordingly, another aspect of the presentinvention provides a method for the gastrointestinal delivery of atherapeutic composition to a patient in need thereof, in which acomposition containing a therapeutic agent with poor gastrointestinalabsorbtion and a peptide ligand according to the present invention isadministered to the gastrointestinal tract of the patient. According toone embodiment the composition is an oral dosage form that isadministered orally. In a further embodiment the composition maycomprise a ligand peptide of the present invention bound to atherapeutic agent or a carrier. In a further embodiment the compositioncomprises at least one peptide ligand, at least one agent and at leastone carrier.

Additional embodiments and advantages to the instant invention will bereadily apparent to one of skill in the art, based on the disclosureprovided herein.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1. Compares the translocation efficiency of peptides #12 (SEQ IDNO. 2) and #19 (SEQ ID NO. 1) with controls.

FIG. 2. Illustrates that peptide-labeled #12 (SEQ ID NO. 2) and itssequence-scrambled peptide FITC-12C1 were transported across Caco-2intestinal epithelial monolayer in the presence and absence ofchylomicron lipid-forming micelles.

FIG. 3. Illustrates a comparison of ApoB48 levels in fractions of Caco-2basolateral side medium.

DETAILED DESCRIPTION OF THE INVENTION 1. Overview

The instant invention relates to the discovery of peptide ligands thatcan be used to transport active agents that are otherwise poorlyabsorbed gastrointestinally (e.g. macromolecular drugs and/or smallmolecules or other therapeutically useful compounds) across theintestinal epithelial cell barrier in a chylomicron-forminglipid-dependent manner. The identified peptide ligands can be bound to acarrier and/or a therapeutic agent.

2. Definitions

As used herein, the singular forms “a,” “an” and “the” include pluralreferences unless the content clearly dictates otherwise.

The term “about”, as used here, refers to +/−10% of a value.

The term “amino acid” refers to natural and/or unnatural or syntheticamino acids, including glycine and both the D and L optical isomers,amino acid analogs (for example norleucine is an analog of leucine) andpeptidomimetics.

As used herein “conservative substitutions” refers to changes that cangenerally be made to an amino acid sequence without altering activity,an amino acid belonging to a grouping of amino acids having a particularsize or characteristic can be substituted for another amino acid.Substitutes for an amino acid sequence may be selected from othermembers of the class to which the amino acid belongs. For example, thenonpolar (hydrophobic) amino acids include alanine, leucine, isoleucine,valine, proline, phenylalanine, tryptophan, and tyrosine. The polarneutral amino acids include glycine, serine, threonine, cysteine,tyrosine, asparagine and glutamine. The positively charged (basic) aminoacids include arginine, lysine and histidine. The negatively charged(acidic) amino acids include aspartic acid and glutamic acid. Exemplaryconservative substitutions include, but are not limited to, Lys for Argand vice versa to maintain a positive charge; Glu for Asp and vice versato maintain a negative charge.

The term “hydrophobic” refers to a substance, molecule, or a domain of asubstance of molecule that does not dissolve or is not wetted by water.The energetic interaction between a hydrophobic substance, molecule, ordomain is unfavorable. Examples include solvents such as hydrocarbonsand esters that when mixed with water undergo phase separation.

The term “hydrophilic” refers to a property wherein a substance, amolecule, or a domain of a substance or molecule has an affinity forwater or aqueous fluids. A hydrophilic substance, molecule, or domaincan dissolve, become deliquescent, or be wetted by water or the aqueoussubstance. A substance, molecule, or a domain thereof is hydrophilicwhen the energetics of the interaction between the substance, molecule,or domain and water or an aqueous fluid is favorable.

As used herein, a “spacing group” or “spacer sequence” refers to aportion of a chemical structure, which connects two or moresubstructures such as amino acids, labels, polymers, through afunctional group. For example a spacer sequence may be an amino acidsequence consisting of at least 2 amino acids. The atoms of a spacinggroup and the atoms of a chain within the spacing group are themselvesconnected by chemical bonds.

As used herein, the term “linker” refers to a chemical moiety thatconnects a molecule to another molecule, covalently links separate partsof a molecule or separate molecules. The linker provides spacing betweenthe two molecules or moieties such that they are able to function intheir intended manner. Examples of linking groups include peptidelinkers, enzyme sensitive peptide linkers/linkers, self-immolativelinkers, acid sensitive linkers, multifunctional organic linking agents,bifunctional inorganic crosslinking agents and other linkers known inthe art. The linker may be stable or degradable/cleavable.

As used herein the term “therapeutic agent” encompasses pharmaceuticallyactive therapeutic agents, diagnostic, biologic and targeting agents, aswell as adjuvants.

As used herein, the term “diagnostic agent” refers to any molecule whichproduces, or can be induced to produce, a detectable signal. Thediagnostic agent may be any diagnostically useful compound that may bebound via a functional group thereon to the composition of theinvention. Diagnostic moieties having reporter molecules that can bedetected by imaging equipment may include radioactive, paramagnetic,fluorescent or radioopaque chemical entities. Non-limiting examples oflabels include radioactive isotopes, enzymes, enzyme fragments, enzymesubstrates, enzyme inhibitors, coenzymes, catalysts, fluorophores, dyes,chemiluminescers, luminescers, or sensitizers; a non-magnetic ormagnetic particle, iodinated sugars that are used as radioopaque agents,and can be appended to linker backbones using ester or other linkages.

As used herein, the term “biologic agent” encompasses substancesgenerally derived from a biological source, such as from an organism, acell line, an isolated tissue, or the like which includes proteins,peptides, carbohydrates, polysaccharides, nucleic acid molecules,examples include antibodies and fragments thereof (including humanized,single chain, chimeric antibodies, fab regions, fc regions,complementarity determining regions, and any combination thereof),peptides (including hormones, for example insulin), and nucleic acidmolecules (including DNA, RNAs, microRNAs, siRNAs, shRNAs, and tRNAs,antisense, aptamers, ribozymes, external guide sequences forribonuclease P, and triplex forming agents).

As used herein the term “nucleic acid” refers to a molecular entitycomposed of a nucleobase, sugar moiety, and phosphate group, or analogsthereof. Examples include the DNA nucleotides, i.e., adenine, guanine,cytosine, and thymidine, or the RNA nucleotide uracil, or syntheticanalogs thereof. Examples of sugar moieties to which the nucleobases arecovalently bonded include but are not limited to ribose and deoxyribose.Analogs of sugars can also be present; for example, halodeoxyriboseanalogs.

As used herein the term “peptide” is used interchangeably with the term“protein” and “amino acid sequence”, in its broadest sense refers to acompound of two or more subunit amino acids, amino acid analogs orpeptidomimetics.

As used herein, the term “amino acid” refers to natural and/or unnaturalor synthetic amino acids, including glycine and both the D and L opticalisomers, amino acid analogs (for example norleucine is an analog ofleucine) and peptidomimetics.

As used herein, the term “conservative substitution” refers to a changethat can generally be made to an amino acid sequence without alteringactivity, an amino acid belonging to a grouping of amino acids having aparticular size or characteristic can be substituted for another aminoacid. Substitutes for an amino acid sequence may be selected from othermembers of the class to which the amino acid belongs. For example, thenonpolar (hydrophobic) amino acids include alanine, leucine, isoleucine,valine, proline, phenylalanine, tryptophan, and tyrosine. The polarneutral amino acids include glycine, serine, threonine, cysteine,tyrosine, asparagine and glutamine. The positively charged (basic) aminoacids include arginine, lysine and histidine. The negatively charged(acidic) amino acids include aspartic acid and glutamic acid. Exemplaryconservative substitutions include, but are not limited to, Lys for Argand vice versa to maintain a positive charge; Glu for Asp and vice versato maintain a negative charge.

“Identical” or “identity” as well as “homology” or “homologue” as usedherein in the context of two or more nucleic acids or peptide sequences,means that the sequences have a specified percentage of nucleotides oramino acids that are the same over a specified region. The percentagemay be calculated by comparing optimally aligning the two sequences,comparing the two sequences over the specified region, determining thenumber of positions at which the identical residue occurs in bothsequences to yield the number of matched positions, dividing the numberof matched positions by the total number of positions in the specifiedregion, and multiplying the result by 100 to yield the percentage ofsequence identity. In cases where the two sequences are of differentlengths or the alignment produces staggered end and the specified regionof comparison includes only a single sequence, the residues of singlesequence are included in the denominator but not the numerator of thecalculation. When comparing DNA and RNA, thymine (T) and uracil (U) areconsidered equivalent. Identity may be performed manually or by usingcomputer sequence algorithm such as BLAST or BLAST 2.0.

“Substantially identical” as used herein refers to that a first andsecond sequence are at least 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%,97%, 98% or 99% identical over a region of 8, 9, 10, 11, 12, 13, 14, 15,16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 30, 35, 40, 45, 50 or morenucleotides or amino acids, or with respect to nucleic acids, if thefirst sequence is substantially complementary to the complement of thesecond sequence. Preferably, such variant nucleic acid and peptidesequences will share 75% or more (i.e. 80, 85, 90, 95, 97, 98, 99% ormore) sequence identity with the sequences recited in the application.Preferably such sequence identity is calculated with regard to the fulllength of the reference sequence (i.e. the sequence recited in theapplication).

“Substantially complementary” as used herein refers to that a firstsequence is at least 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 97%, 98% or99% identical to the complement of a second sequence over a region of 8,9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 30,35, 40, 45, 50 or more nucleotides, or that the two sequences hybridizeunder stringent hybridization conditions.

As used herein, “targeting agents” refer to ligands, polymers, proteins,cytokines, chemokines, peptides, nucleic acids, lipids, saccharides orpolysaccharides, small molecules or any combination thereof, (forexample a gylcolipid, glycoprotein etc) that bind to a receptor or othermolecule on the surface of a targeted cell. An exemplary small-moleculetargeting compound is folate, which targets the folate receptor. Thesetargeting agents may be in addition to the peptide sequences disclosedwhich facilitate transport across the intestinal epithelium. The degreeof specificity can be modulated through the selection of the targetingmolecule. For example, antibodies are very specific. These can bepolyclonal, monoclonal, fragments, recombinant, or single chain, many ofwhich are commercially available or readily obtained using standardtechniques.

Additional examples of targeting agents include RGD peptide, EGFpeptide, DV3 (LGASWHRPDKC) peptide, a LYP peptide (CGNKRTRGC), amembrane-binding domain of IGFBP3 (QCRPSKGRKRGFCW), fMLF, mannose,transferrin ligand and monoclonal anti-bodies, including the drugconjugated derivatives of the above mentioned agents.

As used herein, the term “carrier” can be a liquid phase carrier or asolid phase carrier, e.g., gel, nanoparticle, microparticle, deliveryvehicle, a phage or a virion. A carrier may be further modified byattachment of one or more different molecules, such as additionaltargeting and/or attachment molecules, and/or therapeutic, diagnostic,targeting or biologic agents. The agents may be incorporated into, onto,or coupled to a carrier.

As used herein, the term “nanoparticle” (NP) refers to a structurehaving at least one dimension between about 1-1,000 nm in one or moredimensions. Exemplary nanoparticles include but are not limited todendrimers, liposomes, semiconductor crystals (e.g., quantum dots),metal particles, magnetic particles, carbon tubes, Bucky balls, quantumrods (QRs), quantum wires (QWs), and other nanoparticles.

As used herein, the term “liposome” refers to a lipid vesicle composedof concentric phospholipid bilayers which enclose an aqueous interior(Gregoriadis, et al., hit J Pharm 300, 125-30 2005; Gregoriadis andRyman, Biochem J 124, 58P (1971)). The lipid vesicles comprise eitherone or several aqueous compartments delineated by either one(unilamellar) or several (multilamellar) phospholipid bilayers (Sapra,et al., Curr Drug Deliv 2, 369-81 (2005)).

As used herein, the term “dendrimer” encompasses polymers distinguishedby their repeated branching structure emanating from a central core.

As used herein, a “microparticle” (MP) refers to a particle having anaverage diameter on the order of micrometers (i.e., between about 1micrometer and about 1 mm), while a “nanoparticle” is a particle havingan average diameter on the order of nanometers (i.e., between about 1 nmand about 1 micrometer. The particles may be spherical or non-spherical,in some cases. Nanoparticles and microparticles are jointly referred toherein as particles unless otherwise specified.

As used herein, the terms “polymer,” “polymeric” and similar terms havethe usual meaning known to those skilled in the art and thus may be usedto refer to homopolymers, copolymers (e.g., random copolymer,alternating copolymer, block copolymer, graft copolymer) and mixturesthereof.

As used herein, the term “block copolymer” refers to a polymer composedof two or more different chemical types of monomer units, whereinmonomer units of one type are largely associated only with each other inparticular domains, “blocks,” of the polymer and monomer units ofanother type are also largely associated only with each other in otherparticular domains or blocks of the polymer. The backbone or continuousmolecular chain of the polymer contains domains of at least two blocks.

As used herein, “PEG” is used herein as an abbreviation for polyethyleneglycol. PEGs are included within the broader class of polyalkyleneoxides, which include PEG as well as polypropylene glycols, andpolyglycol copolymers. PEG can have a range of molecular weights. ThePEG molecular weight range contemplated for use in the present inventionis from about 1000 to about 100,000 Da. PEG can be linear, branched,multi-arm, or a combination of branched and multi-arm. Various PEGs canbe derivatized with various groups, such as activated ester (N-hydroxysuccinimidy ester, for example), p-nitrophenyl, aldehyde, amine, thiol,activated thiol (thiopyridine activated thiol, for example), vinylsulfone, maleimide, aminooxy, hydrazine, tosyl, azide, alkyne,cyclooctyne and idoacetamide.

As used herein, “intestinal epithelial barrier” refers to eptithelialcells that line the intestine of an animal. An intestinal eptithelialbarrier can be provided in vitro or in situ, or is located within theintestines of an animal. A non-limiting example of an intestinalepithelial barrier in vitro is a monolayer of Caco-2 cells in a solidsupport such as a petri dish.

“Animal” includes all vertebrate animals including humans. Inparticular, the term “vertebrate animal” includes, but not limited to,mammals, humans, canines (e.g., dogs), felines (e.g., cats); equines(e.g., horses), bovines (e.g., cattle), porcine (e.g., pigs), mice,rabbits, goats, as well as in avians. The term “avian” refers to anyspecies or subspecies of the taxonomic class ava, such as, but notlimited to, chickens (breeders, broilers and layers), turkeys, ducks, agoose, a quail, pheasants, parrots, finches, hawks, crows and ratitesincluding ostrich, emu and cassowary.

3. Peptide Ligands

The present invention provides peptide ligands. The peptide ligands ofthe instant invention include an amino acid sequence of 8-20 aminoacids. Such sequences include at least one pair of amino acids ofopposite charge, which are separated by an amino acid spacer of 1-12amino acid residues containing at least one hydrophobic amino acid. Forexample, in one embodiment, the first member of the pair of amino acidsis negatively charged. Such amino acids include, but are not limited to,aspartic acid (D) or glutamic acid (E). The second member of the pair ofamino acids is positively charged. Such amino acids include, but are notlimited to, lysine (K), arginine (R), or histidine (H). The spacerelement includes at least one hydrophobic amino acid, which includes,but is not limited to, alanine (A), isoleucine (I), leucine (L),methionine (M), phenylalanine (F), tryptophan (W), tyrosine (Y), valine(V), and combinations thereof.

The peptide ligand sequences of the instant invention also may includemultiple pairs of oppositely charged amino acids separated by a singleor multiple spacers containing one or more hydrophobic amino acids oramino acid sequences. To this end, such multiple pairs may be providedin juxtaposition or in a nested relationship.

In certain non-limiting embodiments, the pair(s) of oppositely chargedamino acids are flanked with one or more non-charged or hydrophobicamino acids or sequences of non-charged amino acids, hydrophobic aminoacids or a combinations thereof. Such amino acids may be provided at theN-terminus and/or C-terminus of the oppositely charged amino acid pairor the peptide ligand sequence. Examples of non-charged amino acidsinclude, but are not limited to, serine (S), threonine (T), asparagine(N), glutamine (Q) and, in certain instances, cysteine (C), glycine (G),and proline (P) valine (V), isoleucine (I), leucine (L), tryptophan (W)and proline (P). Hydrophobic amino acids may be any amino acid asdefined herein. In other related embodiments, the described inventionprovides amino acid variants of the identified ligand peptide sequences,SEQ ID NOs: 1-5. These amino acid variants have at least 70%, at least75%, at least 80%, at least 85%, at least 90%, at least 95%, at least96%, at least 97%, at least 98%, or at least 99%, or greater, sequenceidentity compared to a amino acid sequence of this invention, asdetermined using the methods described herein, (for example, BLASTanalysis using standard parameters). One skilled in this art willrecognize that these values can be appropriately adjusted to determinecorresponding identity of proteins encoded by two nucleotide sequencesby taking into account codon degeneracy, amino acid similarity, readingframe positioning, and the like. In other related embodiments, thedescribed invention includes the following formulas, Formulas 1-5, foramino acid variants of the identified ligand peptide sequences.

A. Formula 1

An embodiment of the present invention may be represented by thefollowing Formula 1:X₁B₁Z₁Z₂Z₃B₂X₂,wherein X₁-X₂ are not charged or hydrophobic amino acids, Z₁-Z₃ is aspacer sequence of at least one hydrophobic amino acid, and B₁ and B₂are members of said at least one pair and have opposite charge.

A non-limiting example of such an amino acid sequence includes, but isnot limited to, TKWPVD (SEQ ID NO: 4). The peptide ligands of theinstant invention may also include variants or homologues of SEQ ID NO:4, which may be at least 75% identical to SEQ ID NO: 1 or which maycontain conservative substitutions of corresponding amino acid residues.

B. Formula 2

An embodiment of the present invention may be represented by thefollowing Formula 2:X₁B₁Z₁Z₂Z₃B₂X₂X₃X₄,wherein X₁-X₄ are not charged or hydrophobic amino acids, Z₁-Z₃ is aspacer sequence of at least one hydrophobic amino acid, and B₁ and B₂are members of said at least one pair and have opposite charge.

A non-limiting example of such an amino acid sequence includes, but isnot limited to, TKWPVDMCP (SEQ ID NO: 5). The peptide ligands of theinstant invention may also include variants or homologues of SEQ ID NO:4, which may be at least 75% identical to SEQ ID NO: 1 or which maycontain conservative substitutions of corresponding amino acid residues.

C. Formula 3

An embodiment of the present invention may be represented by thefollowing Formula 3:X₁B₁Z₁Z₂Z₃B₂X₂X₃X₄X₅X₆X₇,wherein X₁-X₇ are not charged or hydrophobic amino acids, Z₁-Z₃ is aspacer sequence of at least one hydrophobic amino acid, and B₁ and B₂are members of said at least one pair and have opposite charge.

A non-limiting example of such an amino acid sequence includes, but isnot limited to, TKWPVDMCPNVS (SEQ ID NO: 1). The peptide ligands of theinstant invention may also include variants or homologues of SEQ ID NO:1, which may be at least 75% identical to SEQ ID NO: 1 or which maycontain conservative substitutions of corresponding amino acid residues.

D. Formula 4

An embodiment of the present invention may be represented by thefollowing Formula 4:X₁B₁B₂Z₁Z₂Z₃Z₄Z₅B₃Z₆Z₇B₄,wherein X₁ is either an uncharged amino acid or hydrophobic amino acid,Z₁-Z₇ is a spacer sequence of at least one hydrophobic amino acid, andB₁-B₄ are charged amino acids and wherein B₁ and B₂ have an identicalcharge which charge is opposite to the charge of B₃ and B₄.

A non-limiting example of such an amino acid sequence includes, but isnot limited to, QDDVQTWQRQPK (SEQ ID NO: 2). The peptide ligands of theinstant invention may also include variants or homologues of SEQ ID NO:2, which may be at least 75% identical to SEQ ID NO: 2 or which maycontain conservative substitutions of corresponding amino acid residues.

E. Formula 5

An embodiment of the present invention may be represented by thefollowing Formula 5:X₁B₁Z₁Z₂B₂Z₃B₃Z₄B₄Z₅Z₆B₅Z₇B₆X₂X₃X₄,wherein X₁-X₄ represent uncharged or hydrophobic amino acids and Z₁-Z₇represent the spacer sequence of at least one hydrophobic amino acid.B₁-B₆ represent charged amino acids, wherein B₁, B₂ and B₃ have anidentical charge which is opposite to that of B₄, B₅ and B₆.

A non-limiting example of such an amino acid sequence includes, but isnot limited to, GENFEQDWKSLRPHSSN (SEQ ID NO: 3). The peptide ligands ofthe instant invention may also include variants or homologues of SEQ IDNO: 3, which may be at least 75% identical to SEQ ID NO: 3 or which maycontain conservative substitutions of corresponding amino acid residues.

COMPOSITIONS

The present invention provides a composition comprising a peptide ligandof the present invention, and a therapeutic agent or a carrier. Thepeptide ligands can be bound to a carrier or conjugated to at least onetherapeutic, biologic, diagnostic or target agent to transport thecarrier or conjugate across the intestinal epithelium barrier. One withordinary skill in the art will understand how to directly conjugate thepeptide ligands to a therapeutic agent or carrier, or utilize a linkerto bind the peptide ligand to a therapeutic agent or carrier. One ormore peptide ligands, as well as different variants of peptide ligandsmay be bound to the agent or carrier.

In a further embodiment the invention provides a composition comprisingan amino acid sequence of the invention and a therapeutic agent bound tothe same carrier molecule. In related embodiments, multiple agents, aswell as multiple types of agents may be conjugated to the peptide ligandor to the carrier. Also, multiple peptide ligands, and variant peptideligands may be bound to the agent and carrier and all ligands and agentsmay be bound to a common carrier.

A. Carrier

The carrier may be formed of any suitable pharmaceutically acceptable ortherapeutically acceptable material, which are well known. The carriermay comprise of a metal, glass, lipid, protein, polymer or anycombinations thereof.

In a preferred embodiment the carrier is a particle formed frombiocompatible or biodegradable polymers such as polylactic and/orpolyglycolic acids, polyanhydrides, polycaprolactones, polyethyleneoxides, polybutylene terephthalates, starches, cellulose, chitosan,and/or combinations of these. The particles may comprise a hydrogel,such as agarose, collagen, or fibrin.

Non-biodegradable or biodegradable polymers may be used to form theparticles. In the preferred embodiment, the particles are formed of abiodegradable polymer. In general, synthetic polymers are preferred,although natural polymers may be used and have equivalent or even betterproperties, especially some of the natural biopolymers which degrade byhydrolysis, such as some of the polyhydroxyalkanoates. Representativesynthetic polymers include poly(hydroxy acids) such as poly(lacticacid), poly(glycolic acid), and poly(lactic acid-co-glycolic acid),poly(lactide), poly(glycolide), poly(lactide-co-glycolide),polyanhydrides, polyorthoesters, polyamides, polycarbonates,polyalkylenes such as polyethylene and poly-propylene, polyalkyleneglycols such as poly(ethylene glycol), polyalkylene oxides such aspoly(ethylene oxide), polyalkylene terepthalates such as poly(ethyleneterephthalate), polyvinyl alcohols, polyvinyl ethers, polyvinyl esters,polyvinyl halides such as poly(vinyl chloride), poly-vinylpyrrolidone,polysiloxanes, poly(vinyl alcohols), poly(vinyl acetate), polystyrene,polyure-thanes and co-polymers thereof, derivativized celluloses such asalkyl cellulose, hydroxyalkyl celluloses, cellulose ethers, celluloseesters, nitro celluloses, methyl cellulose, ethyl cellulose,hydroxypropyl cellulose, hydroxypropyl methyl cellulose, hydroxybutylmethyl cellulose, cellulose acetate, cellulose propionate, celluloseacetate butyrate, cellulose acetate phthalate, carboxylethyl cellulose,cellulose triacetate, and cellulose sulfate sodium salt (jointlyreferred to herein as “synthetic celluloses”), polymers of acrylic acid,methacrylic acid or copolymers or derivatives thereof including esters,poly(methyl methacrylate), poly(ethyl methacrylate),poly(butylmethacrylate), poly(isobutyl methacrylate),poly(hexylmethacrylate), poly(isodecyl methacrylate), poly(laurylmethacrylate), poly(phenyl methacrylate), poly(methyl acrylate),poly(isopropyl acrylate), poly(isobutyl acrylate), and poly(octadecylacrylate) (jointly referred to herein as “polyacrylic acids”),poly(butyric acid), poly(valeric acid), andpoly(lactide-co-capro-lactone), copolymers and blends thereof. As usedherein, “derivatives” include polymers having substitutions, additionsof chemical groups and other modifications routinely made by thoseskilled in the art.

Examples of preferred biodegradable polymers include polymers of hydroxyacids such as lactic acid and glycolic acid, and copolymers with PEG,polyanhydrides, poly(ortho)esters, polyurethanes, poly(butyric acid),poly(valeric acid), poly(lactide-co-capro-lactone), blends andcopolymers thereof.

Examples of preferred natural polymers include proteins such as albumin,col-lagen, gelatin and prolamines, for example, zein, andpolysaccharides such as alginate, cellulose derivatives andpolyhydroxyalkanoates, for example, polyhydroxybutyrate. The in vivostability of the particles can be adjusted during the production byusing polymers such as poly(lactide-co-glycolide) copolymerized withpolyethylene glycol (PEG). If PEG is exposed on the external surface, itmay increase the time these materials circulate due to thehydrophilicity of PEG.

While not intending to be bound by theory, the dependence of the instantpeptides on chylomicron-forming lipid for absorption suggests a routethrough gut lymphatics. This unique gut lymphatic route, unlike theblood absorption, offers the advantage of bypassing the first-pass liverclearance. It is known that the flow rate of gut lymphatic absorption isonly ˜ 1/500^(th) of that of blood absorption, which offers theadvantage of slow oral absorption if it is so desired. Since degradableor releasable delivery vehicles can be engineered, drugs carried in suchvehicles, particularly NPs or MPs, can be released in the gut laminapropria before entering gut lymphatics for faster blood absorption.Therefore, vehicles utilizing the targeting ligands can be made tunablein the distribution between blood and lymphatic routes and in the speedof absorption.

The size of the carrier may also modify the transport properties acrossthe intestinal epithelium barrier. Gut lymphatics at the capillary endare known to have clefts that allow for one-way entry. It is known thatthe entry rate is inversely related to vehicle size. In a preferredembodiment, the increased micron size of the carrier or particle, willlead to a slower speed. A large sized particle will likely reside in gutlamina propria for days or weeks, offering a depot that slowly andconstantly releases a therapeutic, biologic, or diagnostic agent to becirculated by the lymphatic system. This form of slow release isparticularly useful to deliver potent, low concentrationbiomacromolecules such as interferon and insulin.

One of ordinary skill in the art will appreciate that there are numerousparticle compositions that could be used in the presently described andclaimed invention.

B. Therapeutic Agents

Therapeutic agents may be bound to the peptide ligand of the presentinvention by known methods in the art (e.g., by covalent bond,noncovalent interactions, or expressed as a fusion or chimeric protein).A therapeutic agent or multiple therapeutic agents may be bound to acarrier, as well as multiple types of therapeutic agents. In a furtherembodiment, a therapeutic agent may be bound to a carrier using alinker. For example, BIOCONJUGATE TECHNIQUES (Academic Press; 1stedition, Greg T. Hermanson, 1996) describes techniques for modifying orcrosslinking of biomolecules. For example, a diagnostic agent and apharmaceutically active agent may be bound to a peptide ligand of thepresent invention. In another example, multiple types of agents may bebound to a carrier, such as at least one pharmaceutically active agent,at least one biologic agent, at least one diagnostic agent and at leastone targeting agent, or various combinations thereof.

Examples of pharmaceutically active therapeutic agents include drugcompounds such as anti-infective agents, antibiotics, antibiotics thatmay be used include tetracyclines (e.g. minocycline), rifamycins (e.g.rifampin), macrolides (e.g. erythromycin), penicillins (e.g. nafcillin),cephalosporins (e.g. cefazolin), other beta-lactam antibiotics (e.g.imipenem, aztreonam), aminoglycosides (e.g. gentamicin),chloramphenicol, sulfonamides (e.g. sulfamethoxazole), glycopeptides(e.g. vancomycin), quinolones (e.g. ciprofloxacin), fusidic acid,trimethoprim, metronidazole, clindamycin, mupirocin, polyenes (e.g.amphotericinB), azoles (e.g. fluconazole) and betalactam inhibitors(e.g. sulbactam), minocycline, rifampin, erythromycin, nafcillin,cefazolin, imipenem, aztreonam, gentamicin, sulfamethoxazole,vanco-mycin, ciprofloxacin, trimethoprim, metronidazole, clindamycin,teicoplanin, mupirocin, azithro-mycin, clarithromycin, ofloxacin,lomefloxacin, norfloxacin, nalidixic acid, sparfloxacin, peflox-acin,amifloxacin, enoxacin, fleroxacin, temafloxacin, tosufloxacin,clinafloxacin, sulbactam, clavulanic acid, amphotericin B, fluconazole,itraconazole, ketoconazole, and nystatin, antiviral drugs, acyclovir andacyclovir prodrugs, famcyclovir, zidovudine, didanosine, stavudine,lamivu-dine, zalcitabine, saquinavir, indinavir, ritonavir, n-docosanol,tromantadine, idoxuridine, HIV antiviral agents, HIV proteaseinhibitors, anti-fungal agents include amorolfine, isoconazole,clo-trimazole, econazole, miconazole, nystatin, terbinafine, bifonazole,amphotericin, griseofulvin, ketoconazole, fluconazole and flucytosine,salicylic acid, fezatione, ticlatone, tolnaftate, triacetin, zincpyrithione, sodium pyrithione, NSAIDs, NSAID analogs, steroidalanti-inflammatory drugs, ibuprofen, flurbiprofen, ketoprofen, aclofenac,diclofenac, aloxiprin, aproxen, aspirin, diflunisal, fenoprofen,indomethacin, mefenamic acid, naproxen, phenylbutazone, piroxicam,salicylamide, salicylic acid, sulindac, desoxysulindac, tenoxicam,tramadol, ketoralac, flufenisal, salsalate, triethanolamine salicylate,aminopyrine, antipyrine, oxyphenbutazone, apazone, cintazone, flufenamicacid, clonixerl, clonixin, meclofenamic acid, flunixin, coichicine,demecolcine, allopurinol, oxypurinol, benzydamine hydrochloride,dimefadane, indoxole, intrazole, mimbane hydrochloride, paranylenehydrochloride, tetrydamine, benzindopyrine hydrochloride, fluprofen,ibufenac, naproxol, fenbufen, cinchophen, diflumidone sodium, fenamole,flutiazin, metazamide, letimide hydrochloride, nexeridine hydrochloride,octazamide, molinazole, neocinchophen, nimazole, proxazole citrate,tesicam, tesimide, tolmetin, and triflumidate, oncology agents,indomethacin, sancycline, a sancycline analog, olvanil, an olvanilanalog, retro-olvanil, a retro-olvanil analog, olvanil carbamate,budesonide, a budesonide analog, methylprednisolone, amethylprednisolone analog, dexamethasone, a dexamethasone analog,camptothecin, carboplatin, doxorubicin, paclitaxel, saquinavir mesylate,amprenavir, ritonavir, indinavir, nelfinavir mesylate, tipranavir,darunavir and atazanavir sulfate.

4. Pharmaceutical Compositions

The present invention provides a pharmaceutical composition comprisingthe peptide ligands of the present invention and a therapeutic agent,and may include a pharmaceutically acceptable carrier forgastrointestinal administration, suitable for administration to amammal, fish, bird, preferably a human. To administer the pharmaceuticalcomposition to humans or animals, it is preferable to formulate themolecules in a composition comprising one or more pharmaceuticallyacceptable carriers. The phrase “pharmaceutically or pharmacologicallyacceptable” refers to molecular entities and compositions that do notproduce allergic, or other adverse reactions when administered usingroutes well-known in the art. “Pharmaceutically acceptable carriers”include any and all clinically useful solvents, dispersion media,coatings, antibacterial and antifungal agents, isotonic and absorptiondelaying agents and the like.

Examples of pharmaceutically acceptable carriers or additives includewater, a pharmaceutical acceptable organic solvent, collagen, polyvinylalcohol, polyvinyl-pyrrolidone, a carboxyvinyl polymer,carboxymethylcellulose sodium, polyacrylic sodium, sodium alginate,water-soluble dextran, carboxymethyl starch sodium, pectin, methylcellulose, ethyl cellulose, xanthan gum, gum Arabic, casein, gelatin,agar, diglycerin, glycerin, propylene glycol, polyethylene glycol,Vaseline, paraffin, stearyl alcohol, stearic acid, human serum albumin(HSA), mannitol, sorbitol, lactose, a pharmaceutically acceptablesurfactant and the like. Additives used are chosen from, but not limitedto, the above or combinations thereof, as appropriate, depending on thedosage form of the present invention.

5. Nucleic Acids

The present invention also includes isolated nucleic acid sequencesencoding the ligand peptides. In a further embodiment, the presentinvention provides for isolated nucleic acid sequences that encode SEQID NOS. 1-5. One with ordinary skill in the art can derive the nucleicsequence from the ligand peptide sequences disclosed.

In other related embodiments, the described invention providespolynucleotide variants that encode the ligand peptide sequences, SEQ IDNOs: 1-5, These polynucleotide variants have at least 70%, at least 75%,at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, atleast 97%, at least 98%, or at least 99%, or greater, sequence identitycompared to a polynucleotide sequence of this invention, as determinedusing the methods described herein, (for example, BLAST analysis usingstandard parameters). One skilled in this art will recognize that thesevalues can be appropriately adjusted to determine corresponding identityof proteins encoded by two nucleotide sequences by taking into accountcodon degeneracy, amino acid similarity, reading frame positioning, andthe like.

The present invention provides a vector comprising a nucleic acid of theinvention. The vector may be an expression vector. An expression vectormay comprise additional elements. For example, the expression vector mayhave two replication systems allowing it to be maintained in twoorganisms, e.g., in mammalian or insect cells for expression and in aprokaryotic host for cloning and amplification. For integratingexpression vectors, the expression vector may contain at least onesequence homologous to the host cell genome, and preferably twohomologous sequences which flank the expression construct. Theintegrating vector may be directed to a specific locus in the host cellby selecting the appropriate homologous sequence for inclusion in thevector. The vector may also comprise a selectable marker gene to allowthe selection of transformed host cells.

The present invention provides a host cell comprising a vector of theinvention. The cell may be a bacterial, fungal, plant, insect or animalcell.

The present invention provides kits comprising an amino acid and/ornucleic acid of the invention together with any or all of the following:assay reagents, buffers, probes and/or primers, and sterile saline oranother pharmaceutically acceptable emulsion and suspension base. Inaddition, the kits may include instructional materials containingdirections (e.g., protocols) for the practice of the methods of thisinvention.

6. Method to Deliver a Therapeutic Agent and/or Carrier In Vivo

The present invention provides a method to deliver a therapeutic agentacross the intestinal epithelial barrier in vivo. The examples hereindisclosed exemplify a method of conjugating the peptide ligand of thepresent invention to a therapeutic agent. Accordingly, One with ordinaryskill in the art can bind the peptide ligands of the present inventionto a therapeutic agent of interest, and introduce the conjugate in vivo.

The present invention provides a method to deliver a carrier across theintestinal epithelial barrier in vivo. The examples herein disclosedexemplify a method of conjugating the peptide ligand of the presentinvention to an carrier. Accordingly, One with ordinary skill in the artcan bind the peptide ligands of the present invention to an carrier ofinterest, and introduce the conjugate in vivo.

One with ordinary skill in the art can introduce the peptide ligand ofthe present invention bound to a therapeutic agent and/or carrier to ananimal by gastrointestinal administration. Gastrointestinal embodimentsinclude embodiments in which the peptide ligand of the present inventionbound to a therapeutic agent and/or carrier and formulated forintroduction to an animal in accordance with known methods forgastrointestinal delivery, such as by oral administration, rectaladministration, and the like.

The dosage required depends on the choice of the route ofadministration, preferably oral administration; the nature of theformulation; the nature of the patient's illness; the subject's size,weight, surface area, age, and sex; other drugs being administered; andthe judgment of the attending physician. Variations in the needed dosageare to be expected in view of the variety of agents available and thedifferent efficiencies of various routes of administration. Variationsin these dosage levels can be adjusted using standard empirical routinesfor optimization as is well understood in the art.

Oral dosage forms may include tablets and capsules. In particularexamples, an oral dosage range is from about 1.0 to about 100 mg/kg bodyweight administered orally in single or divided doses, including fromabout 1.0 to about 50 mg/kg body weight, from about 1.0 to about 25mg/kg body weight, from about 1.0 to about 10 mg/kg body weight(assuming an average body weight of approximately 70 kg; values adjustedaccordingly for persons weighing more or less than average). For oraladministration, the compositions are, for example, provided in the formof a tablet containing from about 50 to about 1000 mg of the activeingredient, particularly about 75 mg, about 100 mg, about 200 mg, about400 mg, about 500 mg, about 600 mg, about 750 mg, or about 1000 mg ofthe active ingredient for the symptomatic adjustment of the dosage tothe subject being treated.

In one embodiment, the rectal dosage form is an enema. In anotherembodiment, the rectal dosage form is a suppository. Variations indosage levels can be adjusted using standard empirical routines foroptimization as is well understood in the art.

EXAMPLES

The invention now being generally described, it will be more readilyunderstood by reference to the following examples, which are includedmerely for purposes of illustration of certain aspects and embodimentsof the present invention, and are not intended to limit the invention.

Example 1 Transport Across the Intestinal Epithelial Barrier

Fat is absorbed from the human gut in the form of chylomicrons, whichare formed within intestinal epithelial cells from emulsified fatdroplets absorbed from the intestinal lumen. The intestinal epithelialcells then secrete the chylomicrons to the abluminal (blood) side. Dueto their micron-sizes, chylomicrons cannot enter the blood streamthrough the capillaries but rather they enter the gut lymphatics. In theinstant invention, two lead peptides (SEQ ID NOS. 1 and 2) were isolatedfrom a random 12-mer linear peptide library (size 1.2×10⁹) using T7phage display. These two peptides, when attached to nanoscale phagecarriers, are able to cross Caco-2 cell monolayers in a chylomicronlipid-dependent manner. The Caco-2 monolayer is an in vitro model forthe human intestinal epithelium. Each phage displays one sequence of a12 residue linear peptide at about 10 copies per phage. Therefore, eachpeptide-displaying T7 phage can be viewed as an approximately a 65 nmspherical nanoparticle conjugated with peptides on its surface. As shownin FIG. 1, phages displaying peptides SEQ ID NOS. 1 and 2, cross Caco-2monolayers in a chylomicron lipid-dependent manner. Compared to phagesdisplaying several control peptides, the amount of phage nanoparticlestranslocated from the apical to the basolateral side is three-orders andtwo-orders of magnitude higher for SEQ ID NOS. 1 and 2 phage,respectively. The results demonstrate that nanoparticles with thesepeptides displayed on their surfaces will be absorbed after oraladministration.

Three lead phages displaying peptides were sequenced to deduce the aminoacid sequence of the displayed peptide, from the N-terminus toC-terminus. Their sequences shown below demonstrate that they share astructural feature of one or two ion-bridges formed by one or two pairsof charged residues (bold faced) flanking hydrophobic residues (shadedgray). Consequently, it is deduced that all three are amphipathic, orsurfactant-like. SEQ ID NO. 3 is a phage genome rearrangement productand has 17 residues. SEQ ID NO. 3 is also less effective in crossingCaco-2 monolayers than SEQ ID NO. 1 (the best) and SEQ ID NO. 2 (thesecond best).

(SEQ ID NO. 1) a. #19: TKWPVDMCPNVS (SEQ ID NO. 2) b. #12: QDDVQTWQRQPK(SEQ ID NO. 3) c. #23: GENFEQDWKSLRPHSSN.

Fluorescently labeled peptides according to SEQ ID NOS. 1 and 3 weresynthesized. The peptide according to SEQ ID NO. 1 aggregatedimmediately after being diluted from DMSO stock into an aqueoussolution. The peptide according to SEQ ID NO. 2 did not show thisphenomenon by the naked eye but light aggregation was still detected ina particle analyzer with aggregate size showing a sharp peak at 2 nm.Therefore, the peptide according to SEQ ID NO. 2 aggregate may be viewedas a small nanoparticle displaying some peptides according to SEQ ID NO.2 in correct orientation (i.e., on the surface not buried inside thenanoparticle). Even with this imperfect display, some increase inchylomicron lipid-dependent translocation across Caco-2 monolayers isstill noticeable over a scrambled peptide according to SEQ ID NO. 1(12C1) (FIG. 2). FITC-12 was 4.6-fold more efficiently transported thanthe sequence-scrambled control (FIG. 2). Student t test indicates thatthe difference between the two is statistically significant (n=3,p<0.05).

In addition, phage displaying a peptide according to SEQ ID NO. 1, butnot a negative control peptide, dramatically induces ApoB48 production(FIG. 3). ApoB48 is a marker and component of chylomicron. Caco-2monolayers were incubated with phage displaying the best lead peptide(SEQ ID NO. 1) or a negative phage clone (#15 in FIG. 3). The negativephage clone was randomly picked up among a number of negative phageclones, all showing the same baseline transport rate as the startinglibrary phages, a mixture of phages displaying 1.2 billion differentpeptide sequences. The control shows only base level signal. Thus, thedata indicates that (a) it is the displayed lead peptides, not the phagecapsid protein, that confer a nanoparticle the ability to cross Caco-2monolayer and (b) transport across gut epithelial cells is likelychylomicron-dependent.

The specification is most thoroughly understood in light of theteachings of the references cited within the specification. Theembodiments within the specification provide an illustration ofembodiments of the invention and should not be construed to limit thescope of the invention. The skilled artisan readily recognizes that manyother embodiments are encompassed by the invention. All publications,and patents, cited in this disclosure are incorporated by reference intheir entirety. To the extent the material incorporated by referencecontradicts or is inconsistent with this specification, thespecification will supersede any such material. The citation of anyreferences herein is not an admission that such references are prior artto the present invention.

Those skilled in the art will recognize, or be able to ascertain usingno more than routine experimentation, many equivalents to the specificembodiments of the invention described herein. Such equivalents areintended to be encompassed by the following embodiments.

The invention claimed is:
 1. A peptide ligand comprising an isolatedamino acid sequence selected from the group consisting of SEQ ID NOS: 1,2, and
 3. 2. A composition comprising the amino acid sequence of claim1, a therapeutic agent and a pharmaceutically acceptable carrier.
 3. Thecomposition of claim 2 wherein the amino acid sequence is bound to saidagent or said carrier.
 4. The composition according to claim 1, whereinthe therapeutic agent is a pharmaceutically active, diagnostic,biologic, imaging or targeting agent.
 5. The composition according toclaim 1, wherein said therapeutic agent has poor gastrointestinalabsorption.
 6. The composition according to claim 1, wherein saidcarrier is a nanoparticle or a microparticle.
 7. The compositionaccording to claim 1, wherein said carrier comprises a polymer.
 8. Thecomposition according to claim 1, wherein said carrier is equal to orgreater than 50 nm in diameter.
 9. The composition of claim 7 whereinthe polymer is polyethylene glycol.